S90: Comparative metabolomics and physiology of xylose-fermenting yeast adapted to a dilute acid pretreated lignocellulose-containing waste stream

Waste effluents from the forest products industry are sources of lignocellulosic biomass that can be converted to ethanol by yeast after pretreatment (such as using dilute acid hydrolysis). However, the challenge of improving ethanol yields from a mixed pentose and hexose fermentation of a potentially inhibitory hydrolysate still remains. One method for improving productivity and achieving higher ethanol yields is to adapt yeast to the actual conditions of the hydrolysate. Scheffersomyces (Pichia) stipitis CBS 6054 was adapted to pretreated effluent, using repeated sub-culturing techniques.  Both physiological comparisons and comparative metabolic profiling were employed to evaluate the adapted and unadapted parent strains. The strains were compared based on substrate utilization, maximum ethanol concentration, volumetric productivity, and ethanol yield, measured using an HPLC. Differences in metabolic profiles were analyzed through tandem MS/MS. The adapted strain had an ethanol yield of 0.34 ± 0.01 g_ethanol/g_substrate, a 10-fold improvement. In addition, after growth on xylose (50 g/L) for 72 hours prior to the fermentations, the adapted strain utilized 79.8% of the available substrates compared to 13.6% of the parent. Key changes in both extracellular and intracellular metabolite production were identified during the fermentation of the hydrolysate.  In particular, an accumulation of intermediates in the pentose phosphate pathway was observed in the unadapted strain compared to the adapted strain, which likely contributed to the decrease in the use of xylose.  These results can be used to map the biochemical and molecular mechanism of adaptation of yeast to identify target pathways for metabolic engineering.